Human endogenous retrovirus (HERV) K18 was found to encode superantigens (SAgs) which are characterized by the ability to activate T-cells bearing particular TCR Vp elements. Compared to the well characterized bacterial SAgs, viral SAgs are less understood. The HERV SAgs may be essential for establishing the long-term persistent infection of Epstein-Barr virus (EBV) and for the pathogenesis of type-l diabetes. The K18 SAg represents a unique type of ligand for TCRs, distinct from both conventional peptide antigens and other known SAgs. These HERV SAgs are normally dormant and can be transcriptionally activated by interferon-alpha and by EBV. Compared to other SAgs which only activate specific subsets of T cells, the EBV-activated HERV-K18 SAgs stimulates T cells initially in a Vp13-specific manner, but followed by a rapid polyclonal activation, for which the molecular mechanism is unknown. The crystal structures of bacterial SAgs-receptor complexes revealed that each SAg binds distinctly to the host receptors. We hypothesize that the HERV SAg interacts differently than, and/or has host receptor binding kinetics different from those of, the bacterial SAgs. The long-term objective of the application is to determine the three-dimensional structures of the HERV SAgs and their interactions with TCR and MHC molecules. This will require the availability of relatively high quantities of soluble viral SAgs. However, production of soluble and functional viral SAgs has been a challenging problem. Therefore, we propose to perform the following studies: Aim 1: Development of a highly efficient strategy to express soluble HERV-K18 SAgs using a GFP folding reporter system, in which the GFP fluorescence provides information regarding the folding success or failure of the fusion target protein; Aim 2. Functional characterization and crystallization of soluble variants of the HERV-K18 SAgs and their complexes with TCR and class II MHC molecules. A first round of mutagenesis has generated several K18 SAg variants with enhanced fluorescence, suggesting improved solubility. This R21 application is intended to lay the ground work for a subsequent R01 application. The exploratory/development nature of the current application is consistent with the R21 mechanism. A detailed structural and functional characterization of these HERV SAgs and their complexes with the host receptors will help us understand how viral SAgs perform their biological functions and may shed light on how we manage to sleep with the enemy without getting hurt.